Blood urea nitrogen (bun) sensor

ABSTRACT

A BUN (blood urea nitrogen) sensor containing immobilized carbonic anhydrase and immobilized urease for the in vitro detection of urea nitrogen in blood and biological samples with improved performance and precision characteristics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.11/216,041, filed Sep. 1, 2005, which is a non-provisional of U.S.Provisional Application No. 60/606,436, filed Sep. 2, 2004. Each ofthese applications is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

An apparatus and method for rapid in situ determination of urea inliquid samples that is capable of being used in the point-of-careclinical diagnostic field, including use at accident sites, emergencyrooms, in surgery, in intensive care units, and also in non-medicalenvironments.

BACKGROUND OF THE INVENTION

The invention relates to an apparatus and its method of use fordetermining the presence or concentration of urea in a liquid sample,for example in laboratory autoanalyzers and preferably in single-usedisposable cartridges adapted for conducting diverse real-time or nearreal-time assays of analytes.

In specific embodiments, the invention relates to the determination ofurea in biological samples such as blood, blood components and urine.

Micro-fabrication techniques (e.g. photolithography and plasmadeposition) are attractive for construction of multilayered sensorstructures in confined spaces. Methods for microfabrication of BUN(Blood Urea Nitrogen) sensors, for example on silicon substrates, aredisclosed in U.S. Pat. No. 5,200,051 to Cozzette et al., which is herebyincorporated by reference in its entirety. The sensor comprises asilicon chip with a silver/silver chloride electrode over which is aplasticized polyvinylchloride layer containing the ammonium ionophorenonactin. Over this layer is a layer of a film-forming latex materialcontaining urease. Alternative ammonium ionophores include gramicidin D(Nikolelis and Siontorou; Ammonium ion minisensors form self-assembledbilayer, Anal. Chem. 68, 1735, 1996) and bicyclic peptides (Nowak;Design, synthesis and evaluation of bicyclic peptides as ammoniumionophores; Thesis, Worcester Polytechnic Institute, 2003).

The Cozzette et al., device operates in the standard potentiometricmanner, where the enzyme urease converts urea from the sample toammonium ions, these ions are detected by the ammonium-selectivemembrane covering the electrode. The electrical potential at theelectrode is a logarithmic function of the ammonium concentration andthus the bulk urea concentration. By calibrating the sensor with knownstandards containing urea, the urea concentration in the sample can beestimated.

The enzyme carbonic anhydrase (CA) has been used in a carbon dioxide(pCO₂) sensor, where it was added to the electrolyte layer to acceleratethe CO₂/H₂CO₃ aqueous equilibrium. This use of CA is well known andunrelated to urea sensing, see: Lindskog, S., Henderson, L., Kannan, K.,Liljas, A., Nyman, P., and Strandberg, B.: Carbonic Anhydrase, TheEnzymes 5, 587, 1971.

Botre, C. and Botre, F., (“Carbonic Anhydrase and Urease: AnInvestigation In Vitro on the Possibility of Synergic Action,”)Biochimica et Biophysica Acta, 997, 111-4, (1989), contains support forthere being a physiological linkage between in vivo levels of urea andthe production of CA enzyme activity. Botre teaches that placing anammonia gas-sensing electrode, a carbon dioxide gas-sensing electrode,and a pH sensor into a solution containing either urease, or urease andcarbonic anhydrase. It also teaches the addition of urea and a CAinhibitor (acetazolamide) to the solutions. These experiments areconsistent with predictions based on the law of mass action, which arethat the presence of carbonic anhydrase can influence (increase) therate of hydrolysis of urea by urease, since CA effectively removescarbon dioxide from the system by moving it into the gas phase.

Specifically, the bicarbonate formed by the urease reaction is convertedto carbon dioxide by CA, which then diffuses out of the liquid phase andinto the air. This process has the effect of reducing the back reactionin which ammonium ions plus bicarbonate are converted to urea. Thus thenet effect of the presence of CA in this system is to increase the rateof ammonium ion production.

Regarding the law of mass action, it is well known in the art ofreversible enzymatic reactions, generically A=B+C, to add a reagent Dwhich reacts with C, for the purpose of driving the reversible reactionin the direction of B (A. W. Adamson, A Textbook of Physical Chemistry,Academic Press (New York) 1973, chapter 7). While the present inventionalso seeks to use carbonic anhydrase to influence the reactivity ofurease (UR), Botre does not suggest the present invention for at leastthe following reasons:

Botre is silent on analytical determination of the concentration of ureain a sample, and silent on analytical determination of urea inbiological samples of clinical interest, e.g. a blood sample. Botre isalso silent on the immobilization of UR and CA, as well as onpotentiometric electrodes with immobilized enzymes.

Botre is silent on microfabrication of potentiometric electrodes fordetermining any analyte including urea, and on the use of the enzymes URand CA in a system which does not permit the exchange of carbon dioxidefrom solution to an air space, as in a cartridge of the U.S. Pat. No.5,096,669 incorporated herein their entirety by reference and otheranalytical systems where urea is measured electrochemically.

The concept of differential electrochemical e.g. potentiometric andamperometric, measurement is well known in the electrochemical art, seefor example Cozzette, U.S. Pat. No. 5,112,455 and Cozzette, U.S. Pat.No. 5,063,081, both incorporated herein by reference in their entirety.

U.S. Pat. No. 5,081,063 discloses the use of permselective layers forelectrochemical sensors and the use of film-forming latexes forimmobilization of bioactive molecules, incorporated herein by reference.The use of poly(vinyl alcohol) (PVA) in sensor manufacture is describedin U.S. Pat. No. 6,030,827 incorporated by reference. U.S. Pat. Nos.6,030,827 and 6,379,883 teach methods for patterning poly(vinylalcohol)layers and are incorporated by reference in their entirety.

Gel electrophoresis of a typical commercial urease preparation, as shownin FIG. 14, indicates that prior art BUN sensors would not have includedCarbonic Anhydrase as a significant impurity.

SUMMARY OF THE INVENTION

One object of the invention is to provide a device for detecting urea ina sample, comprising: (a) a sensor to which a sample suspected ofcontaining urea may be brought into contact, the sensor including atleast two enzymes, urease and carbonic anhydrase immobilized on at leasta portion of said sensor, and (b) a detector system for processingsignals from the sensor.

Another object is to provide a sensor membrane comprising: awater-permeable matrix, including at least two enzymes, urease andcarbonic anhydrase.

Another object is to provide a device for detecting urea in a sample,comprising: an electrode coated with a first layer, said first layercomprising plasticized polyvinyl chloride and nonactin, and at least twoenzymes, urease and carbonic anhydrase, immobilized on said first layer.

Another object is to provide a device for detecting urea in a sample,comprising: an electrode coated with a first layer, said first layercomprising plasticized polyvinylchloride and nonactin, and a secondlayer positioned over and contacting at least a portion of said firstlayer, said second layer comprising a water-permeable matrix andincluding at least two enzymes, urease and carbonic anhydrase.

Another object is to provide a microfabricated sensing device fordetecting urea in a sample, comprising: (a) a substantially planarsubstrate having a patterned microelectrode surface, (b) a first layerover at least a portion of said substrate comprising a plasticizedpolymer and nonactin, and (c) a second layer over at least a portion ofsaid first layer, comprising a water-permeable matrix including at leasttwo enzymes, urease and carbonic anhydrase.

Another object is to provide an improved ion-selective sensor fordetection of urea having a layer in which is immobilized an enzyme,urease, the improvement comprising: immobilizing at least one otherenzyme, carbonic anhydrase, in said layer.

Another object is to provide a method for assaying urea in a sample,comprising: contacting a sample suspected of containing urea with asensor having at least one layer and at least two enzymes, urease andcarbonic anhydrase, immobilized in said at least one layer, anddetecting a chemical moiety in proximity to said at least one layer witha sensor selected from the group consisting of an ammonium ion sensor, apH sensor, a carbon dioxide sensor, and a bicarbonate sensor.

Another object is to provide a method for improving the sensitivity of aurea sensor, which sensor comprises a detector and at least one layer inwhich is immobilized an enzyme, urease, the method comprising:immobilizing an effective amount of a second enzyme, carbonic anhydrase,in said at least one layer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Nernst plot of a prior art sensor response, in mV, as afunction of log [urea], in M, over the range of 1 to 90 mM urea, for (−)aqueous and (•) whole blood.

FIG. 2 shows a 0.5 M NaOH pH titration of different Elvace polymer lots(volume=20 mL), indicating buffering capacity at pH 7.5 for lot A. Theremaining Elvace lots do not exhibit buffering capacity at pH 7.5.

FIG. 3 shows internal pH of the urease-containing Elvace layer aftercontact with urea-containing calibrant solution (t<10 s). Samples (t>14s) with high buffer capacity maintain a consistent internal pH. (In thefigure, b.c. refers to the sample buffer capacity).

FIG. 4 shows the response curves to buffered aqueous control fluids(M1=50 mM urea; P1=20 mM urea) using different BUN sensor designs. Top:standard BUN sensor; BUN sensor with denatured urease; BUN sensor withprotein (bovine serum albumin) replacing urease. Bottom: standard BUNsensor; BUN sensor with enzyme layer buffered to 7.4 and aged 1 week;BUN sensor with addition of carbonic anhydrase; BUN sensor with dialyzedurease.

FIG. 5 shows whole blood response curves, day 1, for BUN sensors withmodifications to enzyme layer.

FIG. 6 shows whole blood response curves, after 4 days cartridgeincubation at 40° C., for BUN sensors with modifications to enzymelayer.

FIG. 7 shows buffered aqueous control fluid (M1=50 mM urea) responsecurves, after 4 days incubation at 40° C., for BUN sensors withmodifications to enzyme layer. Modified BUN sensors with additionalcarbonic anhydrase exhibit increased signal.

FIG. 8 is a topological schematic side elevation of a potentiometricblood urea nitrogen (BUN) sensor and reference electrode.

FIG. 9 is the top elevation of the sensor of FIG. 8, showing an array ofdifferent sensors on a single chip.

FIG. 10 is an isometric top view of a sensor cartridge cover.

FIG. 11 is an isometric bottom view of a sensor cartridge cover.

FIG. 12 is a top view of the layout of a tape gasket for a sensorcartridge.

FIG. 13 is an isometric top view of a sensor cartridge base.

FIG. 14 is a silver stained SDS-PAGE protein gel with a molecular weightmarker, commercially available urease, and commercially availablecarbonic anhydrase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention permits rapid in situ determinations of urea usinga cartridge having an array of analyte sensors and means forsequentially presenting a sample and a fluid (amended or not) to theanalyte array. The cartridges are designed to be preferably operatedwith a reading device, such as that disclosed in U.S. Pat. No. 5,096,669to Lauks et al., issued Mar. 17, 1992, or U.S. Pat. No. 5,821,399 toZelin, issued Oct. 13, 1998, which are both hereby incorporated byreference in their respective entireties.

The invention will be discussed primarily in terms of application to the-i-STAT system, e.g., the cartridge and analyzer disclosure in Lauks etal., U.S. Pat. No. 5,096,669. However, one skilled in the art willrecognize that the invention can be used more broadly, for example inother single-use disposable sensor formats, in limited multi-use sensorcartridge formats, and in analyzers having sensors that are used andrecalibrated until they fail and are replaced, or in which a membranecomponent containing the enzyme reagents is replaced. These assayformats are well known commercial alternatives in the electrochemicalsensing art.

In the -i-STAT system a BUN sensor, along with several other sensors,first contacts a calibrant fluid and then a blood sample. The role ofthe calibrant is to provide a single solution that acts as a universalstandard of known concentration for each of the sensors. One skilled inthe art will recognize that selecting its composition will require atrade-off between the competing needs of individual sensors, e.g. interms of buffer salt selection, buffer concentration, pH, ionicstrength, reagent stability and the like.

The BUN assay is useful to physicians assessing the health of theirpatients. It measures the level of urea nitrogen in the blood, which isa waste product of protein metabolism cleared by the kidneys. Therefore,it assesses renal function. Typical BUN values are 8-20 mg/100 ml. Thecondition known as azotemia, i.e. increased BUN levels, can indicateimpaired renal function, congestive heart failure, dehydration, shock,hemorrhage into the gastrointestinal tract, stress, acute myocardialinfarction or excessive protein intake. Alternatively, decreased BUNvalues may indicate liver failure, malnutrition, anabolic steroid use,pregnancy and siliac disease.

Urea, NH₂C(O)NH₂, in whole blood is detected by the -i-STAT BUN sensorin a two-step process. First, the urea is enzymatically converted to theproducts NH₄ ⁺ and HCO₃ ⁻, via a complicated mechanism that is not wellunderstood. There are several different representations of the enzymaticdecomposition of urea found in the literature (Steinschaden, (1997)Sensors and Actuators B44, 365-369; H. Suzuki (2001) Biosensors andBioelectronics 16, 725-733; A. J. Taylor (1992), Ann. Clin. Biochem. 29,245-264; D. M. Jenkins (1999) J. Dairy Sci. 82, 1999-2004). Two typicalrepresentations of the reaction, are given below:

(NH₂)₂CO+H₂O

2NH₃+CO₂  (1)

(NH₂)₂CO+3H₂O

2NH₄ ⁺+HCO₃ ⁻+OH⁻  (2)

The second step in the detection of urea is the potentiometricdetermination of ammonium ion activity by the NH₄ ⁺ ion-selectiveelectrode (ISE). It is acceptable to approximate the activity of NH₄ ⁺to be its concentration. This is well known in the electrochemical art(D. Freifelder, Physical Biochemistry: Applications to Biochemistry andMolecular Biology, 2^(nd) ed., W. H. Freeman (San Francisco) 1982,chapter 4). The BUN sensor response, i.e. change in potential due tochanges in the concentration of NH₄ ⁺, is calibrated at known levels ofurea in blood. The plot of the -i-STAT sensor response curve, mV as afunction of time, thus indicates the concentration of ammonium ionwithin the sensor membrane, which provides an estimate indirectly of theurea concentration in blood.

The urease enzyme reaction can also be detected by sensors for themoieties H⁺, CO₂ and HCO₃ ⁻ because these are produced or consumed inthe reaction. Detection of ammonium ion is preferred because the bloodhas a significant background of H⁺, CO₂ and HCO₃ ⁻, which wouldgenerally require a differential measurement (i.e., taking thedifference between an enzyme-coated sensor and an adjacent bare sensor),while the ammonium background level in blood is comparatively low. Theproduction of ions during the urease reaction also increases theconductivity of the sample, which can be detected with a conductivitysensor. Again, since the sample has a high background conductivity, thisis not the preferred detection method. Sensors for pH, CO₂, HCO₃ ⁻ andconductivity are well known in the electrochemical sensing art.

The conversion of urea to ammonium ion and carbon dioxide or tocarbonium ion affects the local pH of the immobilized urease enzyme. Itis known that urease has a pH optimum around pH 6 to pH 7.5. Deviationsfrom this pH range will reduce enzyme activity, which in turn can affectthe linearity of response of the sensor at the higher substrateconcentrations. Changes of pH in the matrix used to immobilize theenzyme can also have significant effects on enzyme performance. This canbe observed for manufactured sensors, in terms of sensor shelf-life.

Output characteristics of a BUN sensor containing only the ureaseenzyme: The BUN sensor linearity of a prior art design (U.S. Pat. No.5,200,051) is shown in FIG. 1. These are Nernst plots of the sensorresponse, in millivolts (mV), as a function of log [urea], over therange of 1 to 90 mM urea.

The typical clinical reportable range is 1-50 mM (at 50 mM, log[urea]=−1.3). As with all sensors, the response does not quite meet thetheoretical linear behavior, especially at the low end of the analyterange. While the theoretical Nernst slope is about 59 mV/decade atambient temperature, this BUN design exhibits approximately 37 mV/decadeat the high (linear) end. Note that as long as the expected slope for amanufactured batch of sensors is known, it can be programmed into thesoftware that runs the sensor test cycle. This value, rather than thetheoretical value of the slope, is then used in calculations. A detaileddescription of well-known equations and calculations used in theoperation of sensors of this type is given in U.S. Pat. No. 5,112,455,fully incorporated herein by reference.

The present invention will be better understood with reference to thespecific embodiments set forth in the following Examples.

1. Enzyme Immobilization and Sensor Performance

This example deals with ways in which enzymes are immobilized on a basesensor in a manner that retains enzymatic activity. The preferredmaterial is ELVACE®, which is a commercially-available adhesiveconsisting of a water-based emulsion of the copolymerpoly(ethylene)(vinylacetate). It is used as the water-permeable matrix(termed BUN cocktail) for immobilizing the enzymes.

We have found that adding sodium phosphate buffer (pH −6.5, 6.8 and pH7.4) to the BUN cocktail improves its buffering capacity. This resultsin the usable life of the cocktail, i.e. the time from preparation toapplication (e.g. microdispensing it onto the sensor surface), beingincreased from about 16 hours up to about 25 days. Microdispensing, asan application method, is disclosed in jointly owned U.S. Pat. No.5,554,339, fully incorporated by reference.

Using either 50, 75 or 100 mM sodium phosphate buffer at pH 7.4, weobserved that reliable printing was maintained for up to about 25 days,based on visual assessment of the printed material on the sensor andsensor performance.

It is known that urease in enzyme activity assays has significantlyreduced activity at pH values above pH 7.4. Urease enzyme activity at pH8.0 is typically about 50% that of pH 7.0. Secondly, a greater sensorresponse at high [BUN] is observed with the added buffer. The reasonsfor the sensor response improvement are discussed below.

It is well known that jack bean (Canavalia ensiformis) urease activityis both buffer and pH-dependent; the optimal pH for urease in TRISbuffer is between pH 6.0 and pH 8.0 (Cesareo, S., and Langton, S.:Kinetic Properties of Helicobacter-Pylori Ureases Compared with JackBean Urease, FEMS Microbiol Lett 99, 15, 1992). In the prior art -i-STATsystem, the calibrant fluid has a pH of 7.4, however in the localizedregion of the BUN sensor, i.e. the BUN matrix and its diffusion layer,the pH may be elevated to about pH 7.85 due to the production ofhydroxide ion (OH−) from enzymatic degradation of urea in the calibrantfluid by urease in the matrix. When the BUN sensor is contacted with theblood sample, typically at pH 7.4, it is known that pH buffering isdominated by hemoglobin (J. E. Sherwin and B. B. Bruegger in ClinicalChemistry Theory, Analysis and Correlation, ed. L. A. Kaplan and A. J.Pesce, C. V. Mosby (St. Louis), 1989, Chapter 21). This can result in adifferent pH in the region of the BUN sensor. These non-optimal pHvalues in the region of the sensor may affect the product distribution,lower the sensor response and increase variability. It has been foundthat addition of the sodium phosphate buffer to the BUN cocktailsignificantly improves buffering of urease in the matrix, and reducesthe difference in behavior between aqueous calibrant fluids and wholeblood samples.

We have found that adding sodium phosphate buffer (pH 6.0, 6.8 and pH7.4) to the BUN cocktail for microdispensing improves the solubility ofthe enzyme in that buffer.

Another way to decrease the sensor response time (the time to reach 95%of the steady state signal) was discovered by determining if the sensorresponse is rate-limited by the formation of NH₄ ⁺. Carbonic anhydrase(CA) was added to the enzyme matrix to remove bicarbonate/carbon dioxideby forming the carbonate anion (CO₃ ²⁻), thus pulling reaction (1) tothe right (as described above). This significantly decreases the sensorresponse time.

It was also found that there is no substantial difference in responsefor a matrix CA concentration of 0.09 mM and 0.5 mM for aqueous samples,but the higher carbonic anhydrase concentration [CA], did give a flatterresponse curve in whole blood. Furthermore, sensors with carbonicanhydrase and the standard urease matrix concentration in a cocktail of0.07 mM, gave a similar response to sensors with carbonic anhydrase and20% more urease. This is consistent with gains in linearity at the highend of the urea response curve and improved precision with the additionof the carbonic anhydrase (i) to limit the reverse reaction i.e.production of urea from NH₄ ⁺ and HCO₃ ⁻ and (ii) from the additionalbuffering capacity arising from the carbonate anion.

Kinetic studies of the BUN sensor indicate two different rate mechanismsare operative over the analyte range of 1-50 mM urea. At the high end ofthis range, the sensor response is limited by the concentration ofurease, as is indicated in modeling studies after Carr & Bowers (1980)p. 209-210. At the low end it is substrate limited.

Where the object of the invention is to make single-use BUN sensors,i.e. ones that are used with a single blood sample and then discarded,as in the -i-STAT system, many millions of these sensors may bemanufactured annually. In this example, addition of CA and buffer to thematrix may contribute to reduced lot-to-lot variability of manufacturinglots at the high end of the analyte range. The extent to which theurease enzyme concentration, as the limiting step is reduced, also mayassist in better sensor performance.

It is desirable that the preferred embodiment of the improved BUN sensorincorporate the following features, (i) sodium phosphate buffer at pH7.4 is best added to the latex mixture (BUN cocktail) that includes theenzymes as this considerably extends the shelf-life of the mixture, (ii)prior to addition to the mixture, the enzyme urease is best dialyzedagainst sodium phosphate buffer to increase the enzyme kinetics, and(iii) carbonic anhydrase is best added to the mixture to increase theefficiency of the urea conversion to NH₄ ⁺. The following experimentaldata provide support for these conclusions.

The performance of the prior art BUN sensor and new BUN sensor arecompared in the FIGS. 4-7. These figures show a portion of the BUNelectrode response as a function of time. The portion of the response attime<14 s indicates the potentiometric response, with respect to thereference electrode, of the BUN electrode to the calibrant solution. Thesignal response at time>18 s is that of the BUN sensor to the testsample, which may be an aqueous-based, control material or a whole bloodsample. The reference electrode is described in jointly owned U.S. Pat.No. 4,933,048, which is incorporated by reference herein. Otherreference electrodes are well known in the electrochemical art couldalso be used with these BUN sensors. FIG. 4A shows the response curvesto aqueous control materials using different BUN sensor compositions.The standard BUN sensor, based on -i-STAT prior art, is indicated by asolid black line. The long dash indicates a BUN sensor made withdenatured urease. The short dash indicates a BUN sensor made with bovineserum albumin replacing the urease. This graph demonstrates that thesignal is generated by the presence of urea being converted by ureaseenzyme. FIG. 4B shows performance indicated with a solid line for astandard BUN sensor. A short dash shows enzyme cocktail buffered to pH7.4 and aged 1 week before being microdispensed. An intermediate dashindicates a BUN sensor which also includes carbonic anhydrase and thelong dash indicates dialyzed urease. The presence of carbonic anhydraseexhibits an increased signal.

The long dash curve is the BUN cocktail prepared with urease dialyzedagainst 50 mM phosphate buffer. An important feature is the greaterinitial response after the transition from contact with calibrant fluidto a blood sample, suggesting that the urease kinetics are substantiallyincreased, i.e. the urease enzyme activity is enhanced and effects therapid conversion of urea to the product NH₄ ⁺, and approaches the ideal“step function” response curve of theoretical potentiometric sensors.

Addition of carbonic anhydrase (CA) to the BUN cocktail the intermediatedash increases the amount of NH₄ ⁺ converted from urea. In other words,CA increases the efficiency of the conversion of urea to ammonium ion.The implication here is that the sensitivity of the sensor increaseswith the addition of CA.

FIGS. 5-7 are plots from a shelf-life study with sensors stored at aconstant 40° C. after manufacture. Storage at this elevated temperatureprovides a means for simulating an increased rate of degradation ofsensors. This can provide a means for predicting the actual shelf-lifeof sensors stored under more typical conditions, e.g. room temperatureand refrigeration. For manufactured sensors of the type described here,it is necessary to ensure that sensors remain usable for up to about oneyear after the purchase date. Response curves in whole blood and inaqueous fluid (m1) are shown for sensors stored at the elevated storagetemperature for one (d1) and four (d4) days.

Two different manufactured sensor batches (A and B) were used for thestandard condition. Only data for the carbonic anhydrase modifications(at two concentrations, 0.09 mM and 0.5 mM) are shown in FIG. 5.

The first plot shows the whole blood (wb) response (FIG. 5). The keyfeatures of the CA modification are (i) the increased mV response for agiven change in urea concentration, i.e., more efficient conversion ofurea to ammonium ion, and (ii) the “flattened” curve shape between about25 and 40 s, implying improved steady-state operation. The [CA]=0.5 mMsensor appears to give a slightly flatter curve shape than the [CA]=0.09mM sensor. Significantly, both CA sensors show negligible decline inpotential between 25 and 40 s which is an improvement over the standardsensor design where CA is absent.

The sensor behavior after four days (d4) at 40° C. incubation is similarto that at one day (d1) as shown in FIG. 6. Note that the lower responsecompared to d1 is due to different wb samples. An ideal sampledata-collection window (see disclosure of jointly owned U.S. Pat. No.5,112,455) placement for the sensor with CA would be at approximately 30s.

The aqueous fluid response for the BUN sensors at d1 in FIG. 7 issimilar to those shown in FIG. 6. The sensor response to the m1 aqueousfluid after four days at 40° C. incubation is given below. TheCA-containing sensor shows the same increased mV response as for thewhole-blood samples. A comparison of the response curves at a 30 sdata-collection window for the CA-containing sensor shows only a smalldifference between whole-blood and aqueous samples; this is a usefulimprovement over the difference observed between whole-blood and aqueoussamples for the standard prior art sensor.

Data disclosed above show (i) that the prior art BUN sensor isenzyme-limited at high concentrations of urea, however significantimprovements in sensor response can be achieved with the addition ofcarbonic anhydrase, (ii) stability of the enzyme matrix (Elvace) isrelated to the presence of acetic acid and that addition of a bufferingcomponent assists in extending its lifetime for sensor application, e.g.microdispensing, (iii) maintenance of an optimal pH in the BUN sensor iscrucial to ensuring high urease activity and thus maximum BUN sensorperformance and it can be achieved by adding buffer to the matrix, (vi)addition of carbonic anhydrase gives BUN sensors with minimaldifferences in the response curves between whole-blood and aqueoussamples, and (v) addition of carbonic anhydrase to the matrix providessensors with acceptable shelf-life.

The following sections (i) provide a description of the manufacture ofthe preferred embodiment of the new BUN sensor, (ii) disclosealternative matrix materials for UR and CA immobilization, (iii) givedetails on the biochemical properties and sources of UR and CA, (iv)disclose alternative membrane buffering systems to phosphate, and (v)disclose alternative ways of making UR-CA membranes for attachment totraditional electrodes, as the invention is not limited tomicrofabricated sensor applications.

Manufacture of a Preferred Embodiment of the New BUN Sensor

A preferred embodiment of the new BUN sensor is manufactured using acombination of thin-film microfabrication processes and microdispensingtechniques. It comprises a thin film silver-silver chloride indicatorelectrode operating in combination with a thin-film silver-silverchloride reference electrode of the type described in U.S. Pat. No.4,933,048.

A substrate wafer of silicon is overlaid with an insulating layer ofsilicon dioxide, prepared by thermal oxidation. Metal layers oftitanium/tungsten, and then silver are deposited on the silicon dioxidebase wafer, then patterned using photolithographic techniques. Anelectrically insulating layer such as polyimide polymer or additionalsilicon dioxide is then photo-patterned to isolate adjacent sensorcircuitry. The silver-silver chloride indicator electrode (diameter ˜200microns) is prepared from the patterned silver using traditionaltechniques, e.g. electrochemical, chlorine gas plasma and oxidation ofAg⁰ by an inorganic oxidant such as Cr₂O₇ ²⁻ or Fe³⁺ in the presence ofchloride ion.

The remaining layers of the BUN electrode include two thick-filmstructures: (i) a semi-permeable membrane film, comprising an organicpolymer layer (e.g., poly(vinyl chloride)—PVC), and an ammonium ionionophore; and (ii) the outermost biolayer, comprising in thisparticular sensor, a film-forming latex (e.g., poly(vinylacetate-co-vinyl alcohol)) and a sufficient amount of the enzymes ureaseand carbonic anhydrase. These layers are deposited by a microdispensingtechnique as described in jointly owned U.S. Pat. No. 5,554,339, whichis incorporated by reference.

The reference electrode portion of the unit cell may be comprised ofoverlaid structures described in U.S. Pat. Nos. 4,933,048 and 5,200,051,both incorporated by reference.

The thick-film ammonium ion-sensitive structure comprises a poly(vinylchloride) (PVC) binder, tris(2-ethylhexyl)phosphate as a plasticizer,and nonactin as the ionophore. The indicator electrode can be madeselective for different ions by using the same binder and plasticizercomposition but with different ionophores. For example, valinomycin,monensin and (methyl)monensin, and tridodecylammonium chloride have beenused to make potassium, sodium, or chloride-ion selective electrodes,respectively. Other ionophores may include, but are not limited to crownethers, trialkylamines, or phosphate esters, and the like.Alternatively, other polymeric binder materials may be used besides PVC.These polymers may include, for example, silicon rubber,polytetrafluoroethylene plastics, or derivatives of PVC containingionizable functional groups (e.g., carboxylates). Other plasticizerssuitable for use in the present invention may include, but are notlimited to tris(2-ethylhexyl)phosphate, nitrocymene, 2-nitrophenyloctylether, dibutyl sebacate, diethyl adipate, phthalates, propylenecarbonate, 5-phenylpentanol, or mixtures thereof. Still other bindersand ionophore combinations may occur to those skilled in the art, whichare within the scope of the present invention. The resultingsemi-permeable ion-selective film may have a thickness in the range ofabout 2 microns to about 200 microns, preferably about 10 to about 30microns.

At this point, it is important to distinguish between the properties ofparticle latices and their film-forming counterparts. A particle latexcomprises a solid polymeric structure, such as polystyrene, which iscoated with a hydrophilic material that allows the polymer particle tobe waterborne. Particle latex materials have been used traditionally toimmobilize all manner of biologically active materials including enzymes(See, Kraemer, D. et al., U.S. Pat. No. 4,710,525). Particle latexescoated with CA and UR may be used in the present invention.

By contrast, a film-forming latex is a colloidal solution comprised of amobile polymeric liquid core, such as a vinyl acetate, with ahydrophilic outer coating. Such a film-forming latex is made by anemulsion-polymerization process in which a water-immiscible organicmonomer or a mixture of monomers is added to an aqueous mediumcontaining a free radical catalyst. The polymerization may be initiated,for example, by mechanical agitation (See, for example, Vanderhoff, J.W., J. Poly. Sci. Polymer Symposium 1985, 72, 161-198). When thismaterial is dried the particles coalesce to form a film which cannot beredispersed in water. Because film-forming latices are water-based andcontain both hydrophilic and hydrophobic components, one may speculatethat these compositions are able to provide a stabilizing environmentfor biologically active species, e.g. enzymes including CA and UR, andconstitute an effective medium for the immobilization or incorporationof the same.

It has further been found that film-forming latices from both naturaland synthetic sources are of significant utility. For example, thefollowing synthetic monomers, their chemically-modified analogues,copolymers, or mixtures thereof may be used to make a film-forminglatex: vinyl acetate, ethylene, acrylate or acrylic acid, styrene, orbutadiene. These and many other materials known to those skilled in theart are available commercially from many sources including Reichhold,Air Products, DuPont, Dow Chemical, or Imperial Chemical Company.Natural isoprene-based polymers are also useful and available fromImperial Adhesives and Chemicals, Inc. and from General Latex andChemical Corp. Elvace from Reichhold is used in the preferred embodimentof the BUN sensor.

Moreover, these materials retain their film-forming properties even whennon-latex water-soluble components (e.g., proteins, enzymes,polysaccharides, and hydrocolloids such as agarose, locust bean gum,guar gum, or combinations of hydrocolloids, or synthetic polymers suchas poly(vinyl alcohol), poly(vinyl pyrrolidone), polyacrylamide and thelike) comprise up to about 25% by weight of the solids content. In thisrespect, a significant consideration related to a microfabricationprocess for the production of sensors is that the established filmadheres effectively to a planar substrate even in the presence of largeamounts of additives (i.e., enzymes).

Various methods can be used to define a layer on a planar substrate. Ifa thick layer (about 5 to about 200 microns) is required,microdispensing of a viscous film-forming latex composition (<500Centipoise as measured on a Brookefield RV viscometer) is preferred.However, if a thin layer (about 0.2 to less than about 5 microns) isrequired, a composition with a lower viscosity is used which can bemicrodispensed directly onto the indicator electrode, or alternatively,either microdispensed or spin-coated onto a positive resist layer (e.g.,Shipley AZ 1370 SF) which has been patterned to leave the area over theindicator electrode exposed. Any suitable solvent known in the art, suchas n-butylacetate and the like, is then used to lift off the resist,along with the excess latex. A separate technique using a photoresistcap may also be used.

Control of the surface energy may be used beneficially to control thespreading of the microdispensed reagent (and, thus, its dimensionality,such as thickness). A fluorocarbon e.g., carbon tetrafluoride, plasmatreatment of a polyimide layer surrounding the indicator electrodecauses the aqueous based latex to exhibit a high contact angle (i.e.,minimizes spreading and increasing thickness).

To immobilize one or more biologically active species in a latex layerit is possible either to mix the species with the latex prior todeposition or impregnate the layer after deposition. Stability of thebiologically active species, particularly enzymes, can be enhanced byadding a cross-linking agent either before or after deposition. Thesecross-linking agents are well-known in the art and may include suchcompounds as glyoxal, glutaraldehyde, melamine formaldehyde, ureaformaldehyde, and phenol formaldehyde. Other suitable cross-linkingagents may possess at least two functional groups which may includevinyl, carboxyl, anhydride, amine, amide, epoxy, hydroxyl, cyano,isocyanato, thiol, halo, in addition to formyl, and stable combinationsof these functional groups (e.g., a chloroalkylepoxide). These additivescan significantly enhance the wet-strength of the biolayer and extendthe shelf-life of the completed sensor. In almost all instances, one ormore of the biologically active macromolecules listed in the precedingor following sections of this disclosure may be successfully immobilizedusing a film-forming latex such as Elvace or Elmer's Glue.

The porosity of the enzyme matrix can be controlled to a significantextent by incorporating certain additives, such as salts (e.g., sodiumchloride) or sugar alcohols (e.g., mannitol, erythritol, or sorbitol),into the latex mixture prior to deposition. For example, the addition ofsorbitol to the latex formulation (at about 1 g/dL of solution)significantly decreases the time needed for wet-up of a desiccated ureasensor. A shorter wet-up period (see jointly owned U.S. Pat. No.5,112,455) provides, in turn, for a faster response.

In the preferred embodiment, the new BUN sensor is packaged into acartridge of the type disclosed in U.S. Pat. No. 5,096,669, which alsocontains a calibrant solution. It is contained in a calibrant package(CALPAK), which is ruptured during the blood sample analysis. Thetypical sequence of events includes the CALPAK being ruptured and thenthe calibration solution passing over the sensor and wetting up thesensor. Typically, the CALPAK of prior art cartridges contained thefollowing ions, sodium, potassium, calcium, chloride, bicarbonate andalso HEPES buffer, glucose, lactate, urea, creatine and creatinine.

With regard to urea in the CALPAK, as it was discovered that thissubstrate can create a build up of product, which causes feedbackinhibition of the enzyme, it can be removed from the calibration fluid,thereby removing feedback inhibition of the enzyme. The preferredconstitution of the CALPAK for the new BUN sensor should include sodium,potassium, calcium, chloride, ammonium, bicarbonate, HEPES buffer,glucose, lactate, creatine and creatinine. In this embodiment ammoniumions rather than urea calibrate the BUN sensor.

Properties and Sources of Urease (E.C. 3.5.1.5)

An ideal property of urease for this application is that it has a lowresidual level of associated substrate (urea<0.0002 μmol/enzyme unit)and other nitrogenous compounds. Furthermore it should be free ofcontaminating proteases. Specific activities greater than 700 U/mgprotein at 25° C. are ideal characteristics. The enzyme preparationsshould also be of high purity. Other desirable characteristics of ureaseare that the K_(m) of the enzyme be in the range of 1 to 50 (mM),preferably closer to 50 mM and that the V_(max) be greater than 16,000(micromol/ml/min) and also the K_(cat) be 5×10⁵ min⁻¹ or greater.

In the preferred embodiment, the preferred source of urease is Jack Beanurease (E.C. 3.5.1.5) is Genzyme Diagnostics (One Kendall Square,Cambridge, Mass., USA, 02139). Item number 70-1661-01 (K_(m): 9.39 mM;V_(max): 16187 micromol/ml/min; K_(cat): 5.91×10⁵ min⁻¹) Other source ofJack Bean Urease (E.C. 3.5.1.5) include; (i) Sigma-Aldrich Canada Ltd.(2149 Winston Park Drive, Oakville, Ontario, Canada, L6H 6J8) Catalognumber U1500, (ii) Toyobo (Toyobo Building, 17-9, Nihonbashi, Koami-cho,Chuo-ku, Tokyo, 103-8530, Japan) Catalog numbers URH-201 or URH-301 and(iii) Worthington Biochemical Corporation (730 Vassar Ave, Lakewood,N.J., USA, 08701)

Urease enzymes are also available from the following organisms;Deinococcus radiodurans, Pseudomonas syringae (pv. tomato), Streptomycesavermitilis, Streptomyces coelicolor, Sulfolobus tokodaii, Brucellamelitensis, Brucella suis, Alcaligenes eutrophus (Ralstonia eutropha),Actinobacillus pleuropneumoniae (Haemophilus pleuropneumoniae), Bacilluspasteurii, Bacillus sp. (strain TB-90), Bacillus subtilis, Bordetellabronchiseptica (Alcaligenes bronchisepticus), Clostridium perfringens,Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes,Lactobacillus fermentum, Listonella damsela (Vibrio damsela), Morganellamorganii (Proteus morganii), Mycobacterium tuberculosis, Mycobacteriumbovis, Proteus mirabilis, Proteus vulgaris, Rhizobium meliloti(Sinorhizobium meliloti), Staphylococcus aureus (strain Mu50/ATCC700699), Staphylococcus aureus (strain N315), Staphylococcus aureus(strain MW2), Staphylococcus epidermidis, Staphylococcus xylosus,Streptococcus salivarius, Synechocystis sp. (strain PCC 6803),Ureaplasma parvum (Ureaplasma urealyticum biotype 1), Ureaplasmaurealyticum (Ureaplasma urealyticum biotype 2), Yersinia enterocolitica,Yersinia pestis, Yersinia pseudotuberculosis, Actinobacilluspleuropneumoniae (Haemophilus pleuropneumoniae), Bacillus pasteurii,Bacillus sp. (strain TB-90), Bacillus subtilis, Bordetellabronchiseptica (Alcaligenes bronchisepticus), Bordetella parapertussis,Clostridium perfringens, Haemophilus influenzae, Helicobacter felis,Helicobacter heilmannii, Helicobacter mustelae, Helicobacter pylori(Campylobacter pylori), Helicobacter pylori J99 (Campylobacter pyloriJ99), Klebsiella aerogenes, Lactobacillus fermentum, Morganella morganii(Proteus morganii), Mycobacterium tuberculosis, Mycobacterium bovis,Proteus mirabilis, Proteus vulgaris, Rhizobium meliloti (Sinorhizobiummeliloti), Staphylococcus aureus (strain Mu50/ATCC 700699),Staphylococcus aureus (strain N315), Staphylococcus aureus (strain MW2),Staphylococcus epidermidis, Staphylococcus xylosus, Streptococcussalivarius, Synechocystis sp. (strain PCC 6803), Ureaplasma parvum(Ureaplasma urealyticum biotype 1), Ureaplasma urealyticum (Ureaplasmaurealyticum biotype 2), Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Actinomyces naeslundii, Actinobacilluspleuropneumoniae (Haemophilus pleuropneumoniae), Agrobacteriumtumefaciens (strain C58/ATCC 33970), Alcaligenes eutrophus (Ralstoniaeutropha), Anabaena sp. (strain PCC 7120), Bacillus halodurans, Bacilluspasteurii, Bacillus sp. (strain TB-90), Bacillus subtilis, Bordetellabronchiseptica (Alcaligenes bronchisepticus), Bordetella parapertussis,Bordetella pertussis, Bradyrhizobium japonicum, Brucella abortus,Candidatus Blochmannia floridanus, Clostridium perfringens,Corynebacterium efficiens, Corynebacterium glutamicum (Brevibacteriumflavum), Escherichia coli O157:H7, Escherichia coli, Haemophilusinfluenzae, Klebsiella aerogenes, Klebsiella pneumoniae, Lactobacillusfermentum, Morganella morganii (Proteus morganii), Mycobacteriumtuberculosis, Mycobacterium bovis, Proteus mirabilis, Prochlorococcusmarinus (strain MIT 9313), Prochlorococcus marinus subsp. pastoris(strain CCMP 1378/MED4), Prochlorococcus sp. (strain PCC 9511), Proteusvulgaris, Pseudomonas aeruginosa, Pseudomonas putida (strain KT2440),Pseudomonas syringae (pv. tomato), Ralstonia solanacearum (Pseudomonassolanacearum), Rhizobium loti (Mesorhizobium loti), Rhizobiumleguminosarum (biovar viciae), Rhizobium meliloti (Sinorhizobiummeliloti), Rhodobacter capsulatus (Rhodopseudomonas capsulata),Rhodobacter sphaeroides (Rhodopseudomonas sphaeroides), Staphylococcusaureus (strain Mu50/ATCC 700699), Staphylococcus aureus (strain N315),Staphylococcus aureus (strain MW2), Staphylococcus epidermidis,Staphylococcus xylosus, Streptomyces avermitilis, Streptomycescoelicolor, Streptococcus salivarius, Streptococcus thermophilus,Synechococcus elongatus (Thermosynechococcus elongatus), Synechococcussp. (strain WH7805), Synechococcus sp. (strain WH8102), Synechocystissp. (strain PCC 6803), Ureaplasma parvum (Ureaplasma urealyticum biotype1), Ureaplasma urealyticum (Ureaplasma urealyticum biotype 2), Vibrioparahaemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Canavalia ensiformis (Jack bean) (Horse bean),Cryptococcus neoformans (Filobasidiella neoformans), Helicobacterbizzozeronii, Helicobacter felis, Helicobacter heilmannii, Helicobacterhepaticus, Helicobacter mustelae, Helicobacter pylori J99 (Campylobacterpylori J99), Helicobacter pylori (Campylobacter pylori),Schizosaccharomyces pombe (Fission yeast), Glycine max (Soybean).

These enzymes can be purified from their natural sources or from genefragments cloned from these organisms and overexpressed in recombinantclones. Additional information on urease enzymes is updated on the website www.expasy.ch or the NCBI databases.

Properties and Sources of Carbonic Anhydrase (E.C. 4.2.1.1)

An ideal property of Carbonic Anhydrase for this application is that ithas low residual levels of nitrogenous compounds and be free ofcontaminating proteases. Specific activity greater than 2500Wilbur-Anderson units/mg protein at 0° C. is quoted by the vendor.(Wilbur, K. M. and N. G. Anderson, 1948, Journal of BiologicalChemistry, 176: 147-154) The enzyme preparations should be of highpurity.

Other ideal characteristics of Carbonic Anhydrase are a K_(m) valuebetween 1 to 50 mM, V_(max) greater than 50 (microl/ml/min), with anideal level above 10,000 and a K_(cat) value greater than 75, but withan ideal level of greater than 10⁵ min⁻¹.

In the preferred embodiment, the preferred source is bovine CarbonicAnhydrase (E.C. 4.2.1.1) from Sigma-Aldrich Canada Ltd. (2149 WinstonPark Drive, Oakville, Ontario, Canada, L6H 6J8) Catalog number C3934.(K_(m): 1.31 mM; V_(max): −64.4 micromol/ml/min; K_(cat): 76.24 min⁻¹).Another source of Bovine Carbonic Anhydrase (E.C. 4.2.1.1) isWorthington Biochemical Corporation (730 Vassar Ave, Lakewood, N.J.,USA, 08701).

Carbonic Anhydrase enzymes are also available from the followingorganisms: Caenorhabditis elegans, Chlamydomonas reinhardtii, Flayerialinearis, Equus caballus (Horse), Macaca mulatta (Rhesus macaque),Macaca nemestrina (Pig-tailed macaque) Monodelphis domestica(Short-tailed grey opossum), Arabidopsis thaliana (Mouse-ear cress), Bostaurus (Bovine), Gallus gallus (Chicken), Flayeria linearis, Tribolodonhakonensis (Japanese dace), Equus caballus (Horse), Rattus norvegicus(Rat), Caenorhabditis elegans, Bos taurus (Bovine), Ovis aries (Sheep),Arabidopsis thaliana (Mouse-ear cress), Hordeum vulgare (Barley), Pisumsativum (Garden pea), Spinacia oleracea (Spinach), Nicotiana tabacum(Common tobacco), Arabidopsis thaliana (Mouse-ear cress), Hordeumvulgare (Barley), Pisum sativum (Garden pea), Oryctolagus cuniculus(Rabbit), Spinacia oleracea (Spinach), Nicotiana tabacum (Commontobacco), Homo sapiens (Human), Mus musculus (Mouse), Vaccinia virus(strain Copenhagen), Vaccinia virus (strain WR), Variola virus, Flayeriabidentis, Flayeria brownii, Flayeria pringlei, Brachydanio rerio(Zebrafish) (Danio rerio), Anabaena sp. (strain PCC 7120), Dunaliellasalina, Erwinia carotovora, Klebsiella pneumoniae, Methanosarcinathermophila, Neisseria gonorrhoeae, Escherichia coli, Escherichia coliO157:H7, Helicobacter pylori J99 (Campylobacter pylori J99),Helicobacter pylori (Campylobacter pylori), Synechococcus sp. (strainPCC 7942) (Anacystis nidulans R2), Synechocystis sp. (strain PCC 6803),Caenorhabditis elegans, Arabidopsis thaliana (Mouse-ear cress), Medicagosativa (Alfalfa), Synechococcus sp. Zea mays (Maize), Urochloapanicoides (Panic liverseed grass), Porphyridium purpureum, a panicoides(Panic liverseed grass), Mycobacterium bovis, Synechococcus sp. (strainWH8102), Rhodopirellula baltica, Bacillus cereus (strain ATCC 14579/DSM31), Nitrosomonas europaea, Phaseolus vulgaris (Kidney bean) (Frenchbean), Lotus japonicus, Flayeria bidentis, Leptospira interrogans,eutrophus (Ralstonia eutropha), Arabidopsis thaliana (Mouse-ear cress),Gossypium hirsutum (Upland cotton), Riftia pachyptila (Tube worm),Corynebacterium glutamicum (Brevibacterium flavum), Methanosarcina mazei(Methanosarcina frisia), Nicotiana tabacum (Common tobacco), Brucellamelitensis, Salmonella typhimurium Q92KYO, Rhizobium meliloti(Sinorhizobium meliloti) [Plasmid pSymA (megaplasmid 1)], Coccomyxa sp.PA Bacillus halodurans, Gossypium hirsutum (Upland cotton), Solanumtuberosum (Potato) [Chloroplast], Drosophila melanogaster (Fruit fly),Streptomyces coelicolor, Rhodopseudomonas palustris, Gossypium hirsutum(Upland cotton), Glycine max (Soybean), Phaseolus aureus (Mung bean)(Vigna radiata), Anthopleura elegantissima (Sea anemone).

These enzymes can be purified from their natural sources or from genefragments cloned from these organisms and overexpressed in recombinantclones. Additional information on carbonic anhydrase enzymes is updatedon the web site www.expasy.ch or the NCBI databases.

Buffering System for Enzyme Matrix

From the literature, the pH optimum for urease is reported as pH 8.0.(Wall & Laidler, 1953, The Molecular Kinetics of the Urea-Urease System.IV. The Reaction in an Inert Buffer, Archives of Biochemistry andBiophysics, vol 43: 307-311). Our data suggest that a pH less than pH8.0, closer to pH range of 6.5 to 7.4, for our urease preparation givesan optimal enzyme activity.

Preferred buffer for use in the preferred cartridge device is −100 mMsodium phosphate at pH −6.8. Sodium phosphate buffers ranging from 10 to−200 mM can be used in the preferred embodiment, including said buffersin the pH range from about −6.5 to 7.4.

Other buffers useful in this device include potassium phosphate, TRIS(trishydroxymethylaminomethane), e.g. TRIS-H₂SO₄ (Wall & Laidler, 1953),HEPES (Cesareo & Langton, 1992, Kinetic Properties of Helicobacterpylori urease compared with jack bean urease, FEMS Microbiology Letters,vol 99: 15-22), TRIS-HCl buffer and barbitone.

Other buffers not generally recommended for use with urease are thosethat include sulfite, bisulfite and phenylsulfite ions. (G. Fasman & C.Niemann, 1951, A reinvestigation of the kinetics of the Urease-Catalyzedhydrolysis of Urea. I. The activity of urease in the presence of Sodiumand Potassium Phosphate, Journal of the American Chemical Society, vol73: 1646-1650.)

Membranes with Urease and Carbonic Anhydrase for Use inNon-Microfabricated Devices

The present invention is not limited to sensing devices that aremicrofabricated. Traditionally, sensing electrodes are made from glassstructures with membranes held in place over the tip of the structure bymeans of an O-ring or similar fastening component, e.g. a fixtureincorporating an attached membrane where the fixture is capable ofmating with an electrode so as to abut the membrane to the ion-selectiveelectrode surface. The prior art for glass potentiometric urea sensorsis well known and can employ a second, ion-selective electrode (ISE) inconjunction with the enzyme layer to detect either ammonium ions, via anonactin-based ISE, or hydronium ions, via pH ISE. Membranes thatcontain urease and that can be attached to a glass electrode by means ofan O-ring are well known in the art. Similar membranes are also wellknown for enzymes such as glucose oxidase, as in the Yellow SpringsInstruments glucose analyzer. It is well known that instruments of thiskind have re-usable electrodes where the membrane is exposed to a seriesof samples and gives a sample urea concentration value for each.Intermittently or with each sample the membrane is also exposed to oneor more calibrant fluids. Typically a wash fluid is also applied to themembrane between samples.

In the present invention membranes that contain both urease and carbonicanhydrase enzymes may be prepared. This immobilization may be either bycovalent attachment or physical entrapment, in a polymeric media. Suchpolymeric materials include but are not limited to nitrocellulose (asdescribed in U.S. Pat. No. 4,713,165); poly (vinyl alcohol), andcopolymers of poly (vinyl alcohol), polypyrrole, polyvinyl pyridine,polyalkylthiophenes (as described in U.S. Pat. No. 5,858,186) andpolyurethane (as described in U.S. Pat. No. 6,673,565).

The urease and carbonic anhydrase enzymes may also be immobilized,either by covalent attachment or physical entrapment, in a sol-gel, orbovine serum albumin (BSA) cross-linked with glutaraldehyde (as in U.S.Pat. No. 4,927,516). Alternatively, immobilization may be based onantibodies that bind urease and carbonic anhydrase.

The preferred embodiment is a polyurethane membrane prepared as follows:(i) mix pH 7.8 Tris buffer (10 mM) and surfactant (e.g. Pluronic F-68)in a vessel, (ii) add urease and carbonic anhydrase solutions made inthe same buffer, (iii) add Hypol™ isocyanate functionalism polyurethaneprepolymer (Hampshire Chemical Corp., a subsidiary of Dow Chemical) andthoroughly mix, (iv) inject mixture into a membrane mold, and (v) curemembrane and cut into disks for attachment to an ammonium ion-selectiveelectrode.

An alternative embodiment in which urease and carbonic anhydrase areimmobilized in bovine serum albumin (BSA) cross-linked withglutaraldehyde is also provided. A urease and carbonic anhydrase layeris deposited to a thickness of about 1 mm on the surface of an ammoniumion-selective electrode. The enzyme layer is deposited by across-linking process using glutaraldehyde as a cross-linking agent. Thecross-linked structure prevents the urease and carbonic anhydrase fromeluting into a liquid specimen. (Solution A): 15 wt-% bovine serumalbumin was dissolved in a pH 8.0 phosphate buffer solution, and 1.5 gof urease and 0.2 g of carbonic anhydrase was dissolved in 5 mL of theresulting solution. (Solution B): 25% glutaraldehyde aqueous solution.(Solution C): 10% glycine aqueous solution. A wire electrode is dippedinto Solution A, dried for about 1 min, dipped into Solution B and thendried for 1 min. This procedure was repeated until a layer having athickness of about 1 mm was formed on the electrode. The electrode wasthen dipped into Solution C for 1 min, thereby removing unreactedenzyme. This completes the deposition of the urease and carbonicanhydrase layer.

Membranes of the type described here may be attached to any class ofsensor known in the art including potentiometric sensors, amperometricsensors, conductimetric sensors, optical sensors, e.g. fiber optic andwave guide devices, piezoelectric sensor, acoustic wave sensors and thelike. Likewise the combination of urease and carbonic anhydrase can beimmobilized directly onto these sensors by means of chemicalcross-linking reagents or by physical adsorption. The optical sensor mayinclude a dye material that changes optical properties as a function ofconcentration of H+, NH₄+, CO₂ and HCO₃ ⁻.

Additional BUN Sensors

A potentiometric chemical sensor for urea can be viewed as a system,which is constructed from functionally dissimilar components. In oneembodiment of the blood urea nitrogen (BUN) sensor, the outermost layer,the one in contact with the analyte solution, permits the transport ofurea while also serving to immobilize the enzymes urease and carbonicanhydrase. These enzymes catalyze the hydrolysis of urea to ammonia asdescribed above. At neutral pH values, the ammonia thus produced existspredominantly as ammonium ions. By interposing a separate layeredstructure, which contains an ionophore with high sensitivity andselectivity for ammonium ions between the enzyme containing layer and asilver-silver chloride indicator electrode, the ammonium ionconcentration at the electrode interface can be measured. In this typeof measurement, the potential difference between the indicator electrodeand a reference electrode is recorded.

The analytical value of the measurement is derived from the fact thatthe magnitude of the potential difference is related by the Nicolskyequation (Eq. 3, below) to the concentration of the analyte, in thiscase, urea:

E=E _(o) +RT/nF log [A+Σ(a,b)k(a,b)B]  (3)

where E is the measured electromotive force (signal), R is the gas lawconstant, T is the absolute temperature, n is the absolute value of thecharge on analyte species a (e.g., n=1 for the ammonium ion), F is theFaraday constant, A is the activity of the analyte species a, B is theactivity of an interfering chemical species b, k_(a,b) is theinterference coefficient associated with the effect of the presence ofchemical species b on the electrochemical potentiometric determinationof the activity of the analyte species a, and E_(o) is a constantindependent of T, A, or B. For additional discussion of the Nicolskyequation, refer to Amman, D., Ion-Selective Microelectrodes, Springer,Berlin (1986) p. 68 and references cited therein.

In a preferred embodiment of the present invention, the unit cell forthe BUN sensor comprises a thin film silver-silver chloride indicatorelectrode operating in combination with a thin-film silver-silverchloride reference electrode.

Referring now to the topological illustration in FIG. 8, the substratewafer, 20, is silicon, with an overlaid insulating layer of silicondioxide, 15. The first metal layer, 10, is titanium and serves thefunctions of a conductor and an adhesion layer to the wafer. Succeedinglayers 4 and 4′, are the silver and silver chloride layers. On the leftside of FIG. 8, the remaining layers of the indicator electrode include(i) a semi-permeable membrane film, 25, comprising an organic polymerlayer (e.g., poly(vinyl chloride)—PVC) and an ammonium ion ionophore;and (ii) the outermost biolayer, 11, comprising in this particularsensor, a film-forming latex (e.g., poly(vinyl acetate-co-vinylalcohol)) and a sufficient amount of the enzymes urease and carbonicanhydrase.

The reference electrode portion of the unit cell may be comprised ofoverlaid structures as shown in FIG. 8. In this particular embodiment,the metal and chloridized layers of the reference electrode are coveredby an electrolyte layer, 12, which may comprise any material which isable to hold a high concentration of salt but which is, preferably,photoformable. In this respect, a polyvinylalcohol (PVA) formulation isthe preferred material and may first be photopatterned and forms awater-permeable matrix that can subsequently be saturated with a salt,such as potassium chloride. A separate gas permeable membrane, 8′, mayalso be present which serves to diminish the loss of electrolyte or saltto the bulk analytical sample but allows the rapid wet-up (i.e., passageof H₂O or other small gaseous molecules) of the reference electrodeprior to commencing the sample analysis. The photoresist cap 9, whichmay be a remnant of the patterning process need not be removed if itdoes not bar the free passage of solvent, solute, or ions. In apreferred embodiment, the reference electrode structure described inU.S. Pat. No. 4,933,048, incorporated herein by reference, is used.Alternatively, a reference electrode structure can be used in which thedistance between the liquid junction and the surface of thesilver/silver chloride is sufficiently large, such that theconcentration of electrolyte in the immediate vicinity of the Ag/AgClstructure is substantially constant for a period of time sufficient toperform a measurement of the potential difference between the indicatorelectrode and the reference electrode.

As illustrated in FIG. 8, superimposed over the indicator electrode of aBUN sensor is a thick film ammonium ion-sensitive structure comprising apoly(vinyl chloride) (PVC) binder, tris(2-ethylhexyl)phosphate as aplasticizer, and nonactin as the ionophore. The indicator electrode canbe made selective for different ions by using the same binder andplasticizer composition but with different ionophores. For example,valinomycin, monensin and (methyl)monensin, or tridodecylammoniumchloride have been used to make potassium, sodium, or chloride-ionselective electrodes, respectively. Other ionophores may include, butare not limited to crown ethers, trialkylamines, or phosphate esters,and the like. Alternatively, other polymeric binder materials may beused besides PVC. These polymers may include, for example, siliconrubber, polytetrafluoroethylene plastics, or derivatives of PVCcontaining ionizable functional groups (e.g., carboxylates). Otherplasticizers suitable for use in the present invention may include, butare not limited to tris(2-ethylhexyl)phosphate, nitrocymene,2-nitrophenyloctyl ether, dibutyl sebacate, diethyl adipate, phthalates,propylene carbonate, 5-phenylpentanol, or mixtures thereof. Still otherbinders and ionophore combinations may occur to those skilled in theart, which are within the scope of the present invention. The resultingsemi-permeable ion-selective film may have a thickness in the range ofabout 2 .mu.m to about 200 .mu.m, preferably about 10 to about 30 .mu.m.

Referring now to FIG. 9, indicator electrode, 30, and the adjacentreference electrode, 35, are each connected by an overpassivated signalline, 2, to a contact pad, 1. The unit cell is confined within arectangular area, which is repeated in an array several hundred times ona single silicon wafer. In particular embodiments of the instantinvention, other indicator electrodes may be present in the unit cellfor the simultaneous measurement of other species (e.g., Na.sup.+,K.sup.+, or Cl.sup.−) in addition to ammonium ion.

BUN Biolayer

At this point, it is important to distinguish between the properties ofparticle latices and their film-forming counterparts. A particle latexcomprises a solid polymeric structure, such as polystyrene, which iscoated with a hydrophilic material that allows the polymer particle tobe waterborn. Particle latex materials have been used traditionally toimmobilize all manner of biologically active materials (See, Kraemer, D.et al., U.S. Pat. No. 4,710,825). However, an important property of somebut not all particle latices, which is unsuitable in the presentapplication, is that even after these materials have been dried, theparticles can be redispersed easily in water. By contrast, afilm-forming latex is a colloidal solution comprised of a mobilepolymeric liquid core, such as a vinyl acetate, with a hydrophilic outercoating. Such a film-forming latex is made by an emulsion-polymerizationprocess in which a water-immiscible organic monomer or a mixture ofmonomers is added to an aqueous medium containing a free radicalcatalyst. The polymerization may be initiated, for example, bymechanical agitation (See, for example, Vanderhoff, J. W., J. Poly. Sci.Polymer Symposium 1985, 72, 161-198). When this material is dried theparticles coalesce to form a film, which cannot be redispersed in water.Because film-forming latices are water-based and contain bothhydrophilic and hydrophobic components, one may speculate that thesecompositions are able to provide a stabilizing environment forbiologically active species and constitute an effective medium for theimmobilization or incorporation of same.

It has further been found that film-forming latices from both naturaland synthetic sources are of significant utility. For example, thefollowing synthetic monomers, their chemically-modified analogues,copolymers, or mixtures thereof may be used to make a film-forminglatex: vinyl acetate, ethylene, acrylate or acrylic acid, styrene, orbutadiene. These and many other materials known to those skilled in theart are available commercially from many sources including Reichhold,Air Products, DuPont, Dow Chemical, or Imperial Chemical Company.Natural isoprene-based polymers are also useful and available fromImperial Adhesives and Chemicals, Inc. and from General Latex andChemical Corp.

Moreover, these materials retain their film-forming properties even whennon-latex water-soluble components (e.g., proteins, enzymes,polysaccharides such as agarose, or synthetic polymers such aspoly(vinyl alcohol), poly(vinyl pyrrolidone), and the like) comprise upto about 25% by weight of the solids content. In this respect, asignificant consideration related to a microfabrication process for theproduction of biosensors is that the established film adhereseffectively to a planar substrate even in the presence of large amountsof additives (i.e., enzymes).

Various methods can be used to define a layer on a planar substrate. Ifa thick layer (about 5 to about 200 .mu.m) is required, microdispensingof a viscous film-forming latex composition (<500 Centipoise as measuredon a Brookefield RV viscometer) is preferred. However, if a thin layer(about 0.2 to less than about 5 .mu.m) is required, a composition with alower viscosity is used which can be microdispensed directly onto theindicator electrode, or alternatively, either microdispensed orspin-coated onto a positive resist layer (e.g., Shipley AZ 1370 SF)which has been patterned to leave the area over the indicator electrodeexposed. Any suitable solvent known in the art, such as n-butylacetateand the like, is then used to lift off the resist, along with the excesslatex. A separate technique using a photoresist cap may also be used.

Control of the surface energy may be used beneficially to control thespreading of the microdispensed reagent, and thus its dimensionality,such as thickness. A fluorocarbon (e.g., CF₄) plasma treatment of apolyimide layer surrounding the indicator electrode causes the aqueousbased latex to exhibit a high contact angle (i.e., minimizes spreadingand maximizing thickness).

To immobilize one or more biologically active species in a latex layerit is possible either to mix the species with the latex prior todeposition or impregnate the layer after deposition. The stability ofthe biologically active species, particularly enzymes, is usuallyenhanced by adding a crosslinking agent either before or afterdeposition. These crosslinking agents are well-known in the art and mayinclude such compounds as glyoxal, glutaraldehyde, melamineformaldehyde, urea formaldehyde, and phenol formaldehyde. Other suitablecrosslinking agents may possess at least two functional groups, whichmay include vinyl, carboxyl, anhydride, amine, amide, epoxy, hydroxyl,cyano, isocyanato, thio, halo, in addition to formyl, and stablecombinations of these functional groups (e.g., a chloroalkylepoxide).These additives can often significantly enhance the wet-strength of thebiolayer and extend the shelf-life of the completed sensor.

In a particular embodiment of the present invention, a film-forminglatex is used to immobilize the enzymes urease and carbonic anhydrase. Ahigher enzymatic activity is achieved in this case compared to a ureasensor containing urease alone.

The porosity of the enzyme layer (biolayer) can be controlled to asignificant extent by incorporating certain additives, such as salts(e.g., sodium chloride) or sugar alcohols (e.g., mannitol, erythritol,or sorbitol), into the latex mixture prior to deposition. For example,the addition of sorbitol to the latex formulation (1 g/dL of solution)significantly decreases the time needed for wet-up of the desiccatedurea sensor. A shorter wet-up period provides that can give results forblood analyses more rapidly. To manufacture the base sensor, a siliconwafer with a topical layer of silicon dioxide, which had previously beencleaned, scrupulously with a mixture of concentrated sulfuric acid andhydrogen peroxide is placed into a plasma deposition system and layersof titanium (0.1 .mu.m) and silver (0.5 .mu.m) are sputteredconsecutively onto the wafer surface. The silver-titanium bilayer isthen processed to localize it to a region, which in the final deviceacts as the ammonium ion sensor. This process is achieved by a standardlithographic technique in which the wafer is spin-coated with positiveresist (Shipley AZ 1370 SF). After UV exposure of the photoresistthrough a mask and development (Shipley AZ 351), the exposed silver isremoved by an aqueous solution of ferric nitrate (0.9 mM) as theetchant. The underlying titanium layer is then processed by means of thesame photolithographic steps, but using an aqueous mixture of nitricacid (3.9M) and hydrofluoric acid (0.78M) is used as the etchant.N-methylpyrrolidone solvent is then used to remove the remainingphotoresist to expose the required silver structures (diameter about 150.mu.m).

To passivate the signal lines a photo-definable polyimide (DuPont 2703)is spin-coated onto the wafer. Once the wafer is UV exposed anddeveloped with a solvent the polymer is baked in an oven at 350.degree.C. for 30 minutes under an inert atmosphere and left to cool to150.degree. C. before removal.

The silver is then chloridized by dipping the entire wafer into anaqueous solution of potassium dichromate (12 mM) and hydrochloric acid(60 mM). Over these patterned silver chloride electrodes is placed anammonium ion sensitive membrane. The membrane material is made bydissolving low molecular weight PVC (Sigma) and high molecular weightcarboxylated PVC (Type Geon, Goodrich) (1:1 w/w) in a solvent system ofcyclohexanone, propiophenone, and N-methylpyrrolidone (1:1:1 v/v/v) to atotal solids content of 10 g/dL of solution. Dissolution is accomplishedby heating the mixture at 70.degree. C. for 30 minutes. To this mixturethe plasticizer tris(2-ethylhexyl)phosphate (Fluka) is added, to providea total solids content of 35 g/dL. The resulting mixture is then allowedto cool to 45.degree. C. and nonactin (Fluka) is added in the amountequivalent to 2 percent of the total solids in the mixture. At roomtemperature, 10-100 mL of this final material is microdispensed ontoeach of the silver chloride indicator electrodes on the wafer,overlapping on all sides by at least about 30 .mu.m. Curing isaccomplished by placing the wafer on a 60.degree. C. hot-plate for 30minutes. This process yields a stable, rugged structure having athickness of about 15 .mu.m. The wafer is then washed and partiallydiced. The urease solution is prepared by adding 305 mg of Urease(Genzyme Cat #1661 Jack Bean Urease E.C. 3.5.1.5>300 units/mg specificactivity, free ammonia<0.0002 mol/unit) to a sterile plastic tube, towhich 2.20 g of 100 mM sodium phosphate buffer, pH 6.8 was added to thetube and gently mixed on ice. The Carbonic Anhydrase solution isprepared separately with 0.3 g of Carbonic Anhydrase (Sigma-Aldrich CatNo. C3934) added to 1 ml of 100 mM sodium phosphate buffer, pH 6.8 in asterile plastic tube and mixed gently on ice. A PVA solution is preparedby adding 1.4500 g PVA (Polysciences Cat No. 04398 or Aldrich Cat No.36, 310-3) to 27.4900 g of deionized sterile water, which is mixed on astirring plate at 150° C. then allowed to cool to room temperature.

A microdispensing solution is prepared by adding 2.33 g of the ureasesolution with 3.38 g of Latex (ELVACE Reichold Cat No. 40711-00), 0.58mL of the carbonic anhydrase solution, 1.55 g of 100 mM sodiumphosphate, pH 6.8, 1.12 g of PVA solution.

The following formulations can be loaded into a microsyringe assemblyfor the purpose of establishing ion-sensitive layers in a controllablemanner. The microsyringe assembly is preferably equipped with 25 to 30gauge needles (EFD Inc.) having an internal diameter of 150 .mu.m and anexternal diameter of 300 .mu.m. Typically, the microsyringe needle,which includes an elongated member and a needle tip, is made of ametallic material, like stainless steel. Additional layers may be coatedonto the needle to change its surface properties. Furthermore, othermaterials such as synthetic polymers may also be employed inmanufacturing the main body of the needle, itself. Depending on thepretreatment of the electrode surface and the volume amount of fluidapplied, membrane layers of a thickness ranging from about 1 to about200 .mu.m can be obtained consistently.

Automated Microdispensing System

An important aspect of the microfabricating process described in thepresent invention is an automated system, which is able to microdispenseprecise and programmable amounts of the materials used in the sensors ofinterest. The microdispensing system is comprised of a vacuum chuck anda syringe, each of which are attached to separate means for altering oneor more of the vertical, horizontal, lateral, or rotational displacementof these elements. For the sake of economy, it is sufficient to havemeans for changing the vertical displacement of the syringe so long asone can change the position of the vacuum chuck multidirectionally. Themovements of both elements may be controlled via a personal computer.The position of the vacuum chuck may be reproducible within .+−.13microns or better in either x or y directions.

The drop sizes which can be dispensed reproducibly extends over a widerange. For volume sizes between about 5 to about 500 nanoliters (nL),the drops can be applied with a precision of about 5%. A solenoid havinga 0.1% precision rating is sufficient for this purpose. The height ofthe tip of the syringe needle above the sensor should be between about0.1 to about 1 mm, depending on the volume to be dispensed: generally,the smaller the volume of the drop, the lower the elevation of theneedle from the sensor.

The precise alignment of the syringe needle with the preselected area ofthe sensor can be achieved optically by means of a camera and a reticle.Such an operation can be performed manually by an operator orautomatically by means of a visual recognition system.

Volumetric Microdispensing of Fluids

It is useful, at this point, to consider the dynamics involved when asingle drop of fluid is formed at and expelled from a needle

As more fluid is expelled from the needle tip, the drop will grow insize until the gravitational force acting on the mass of the dropexceeds the opposing forces maintaining contact with the needle tip.These opposing forces include the adhesive forces between the needle tipand the fluid or liquid, and surface tension of the liquid itself. It iswell established that at low liquid flow rates where discrete dropformation is complete, the drop volume is fixed. However, the volume maybe changed by varying any of the fluid related parameters discussedabove, or by changing the diameter of the needle tip thus changing theavailable surface area for fluid adhesion. For example, a hydrophopicpolytetrafluoroethylene (PTFE) coating applied to the needle tip reducesthe natural drop size of an aqueous based latex material by reducing theadhesive forces between the drop and the needle tip.

In circumstances where a controlled volume must be microdispensed onto asurface, it is possible to have the microsyringe tip positioned abovethe planar surface at a height which does not allow the drop to formcompletely (and then fall to the surface under the influence ofgravity), but the partially formed drop actually contacts the surfaceand the new adhesive forces between the liquid and the surface begin tospread the drop. If the needle tip is now retracted in the Z-direction asufficient distance away from the surface, then the cohesive forces ofthe liquid is overcome and a volume of liquid less than the fixed dropsize will remain in contact with the surface. This technique can be usedto dispense reproducibly any volume of liquid from about one-onethousandth of the fixed drop size and greater.

Fluid Compositions with Predetermined Surface Tension

The surface tension between a pure liquid and its vapor phase can bechanged by adding reagents. For example, a fatty acid added to waterreduces the surface tension, whereas added salts can increase surfacetension.

The microdispensable fluid compositions of the present invention areprepared to have a controlled optimized surface tension. Suitableadditives are used when necessary. The hydrophobicity or hydrophilicityof the fluid is controlled in the same manner. Where a cured membrane isrequired as the end product, the solids content and volatile solventscontent are carefully adjusted. Moreover, the ratio of these componentsis also used to control the viscosity.

The preferred microdispensable compositions for the ammonium ion sensorcomprises PVC polymer, plasticizers, ionophores and solvents withviscosities generally higher than those used for planar casting (e.g.,spin-coating) of membranes. These higher viscosity compositions cure ordry without deformation of the membrane layer. Related problems, e.g.,that of ensuring the homogeneity of the matrix at high viscosity andthus preventing phase separation of materials after time (i.e.,considerations related to shelf-life) are also alleviated by thesecompositions. Other additives are also used to prevent long-termdegradation of the membranes. Finally, the solvent system is selected toprovide the appropriate surface tension and stability. For NH₄ ⁺sensors, the solids content (wt %) of plasticizer, PVC polymer, andionophore are preferably 60-80%, 15-40% and 0.5-3%, respectively.

Methods for Tailoring the Surface Energy of a Planar Structure

In addition to the factors described above relating to controlledvolumetric dispensing of fluids having an optimized surface tensionassociated with a prescribed composition, tailoring the surface freeenergy of the substrate, or surface onto which the fluid is dispensed,provides control over the final dimensions, especially the thickness, ofthe resulting layer. The resulting process is highly versatile, allowingthe deposition of arrays of layers of varied composition and utility.

For establishing thick membranes, (e.g., 40-60 .mu.m thick), the surfaceis preferably tailored so that the contact angle which themicrodispensed fluid makes with the surface is large. For example,before an aqueous latex membrane is microdispensed, the surface is firstplasma treated with carbon tetrafluoride to yield a contact angle forwater (control fluid) in the range 50°-70°.

Cartridge Construction:

The preferred embodiment provides cartridges and methods of their usefor processing liquid samples to determine the presence or amount of ananalyte in the sample.

Referring to the Figures, the cartridge of the present inventioncomprises a cover (two views), FIGS. 10, 11, a base, FIG. 13, and athin-film adhesive gasket, FIG. 12, disposed between the base and thecover and securing them together. Specifically, the backside of thecover shown in FIG. 10 mates with the exposed face of the gasket of FIG.12, and the backside of the gasket mates with the exposed face of thebase of FIG. 13. Referring now to FIG. 10, the cover 1 is made of arigid material, preferably plastic, and capable of repetitivedeformation at flexible hinge regions 5, 9, 10 without cracking. Thecover comprises a lid 2, attached to the main body of the cover by aflexible hinge 9. In operation, after introduction of a sample into thesample holding chamber 34, the lid can be secured over the entrance tothe sample entry port 4, preventing sample leakage by means ofdeformable seal 11, and the lid is held in place by hook 3. The coverfurther comprises two paddles 6, 7, that are moveable relative to thebody of the cover, and which are attached to it by flexible hingeregions 5, 10. In operation, when operated upon by a pump means, paddle6 exerts a force upon an air bladder comprised of cavity 43, which iscovered by thin-film gasket 21, to displace fluids within conduits ofthe cartridge. When operated by a second pump means, paddle 7 exerts aforce upon the gasket 21, which can deform. The cartridge is adapted forinsertion into a reading apparatus, and therefore has a plurality ofmechanical and electrical connections for this purpose. It should alsobe apparent that manual operation of the cartridge is possible. Thus,after insertion of the cartridge into a reading apparatus, the readingapparatus transmits pressure onto a fluid-containing foil pack filledwith approximately 130 uL of calibrant fluid located in cavity 42,rupturing the package upon spike 38, and expelling fluid into conduit39, which is connected via a short transecting conduit in the base tothe sensor conduit, 12. When the calibrant fluid contacts the sensors,they wet-up and establish a signal associated with the amount ofcalibrating ion or molecule in the fluid.

Referring to FIG. 12, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Holes 30 and 33 permit one or more urea sensors and one ormore reference electrode that are housed within either cutaway 35 or 37,to contact fluid within conduit 12.

Referring to FIG. 13, conduit 34 is the sample holding chamber thatconnects the sample entry port 4 to first conduit 15 in the assembledcartridge. Cutaways 35 and 37 optionally houses a conductimetric sensorfor determining the position of air-liquid boundaries. Recess 42 housesa fluid-containing package, e.g., a rupturable pouch, in the assembledcartridge that is pierced by spike 38 because of pressure exerted uponpaddle 7 upon insertion into a reading apparatus. Fluid from the piercedpackage flows into the second conduit at 39 and then into conduit 12. Anair bladder is comprised of recess 43 which is sealed on its uppersurface by gasket 21. The air bladder is one embodiment of a pump means,and is actuated by pressure applied to paddle 6 which displaces air inconduit 40 and thereby displaces the sample from sample chamber 34 intoconduit 15 and then 12.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

1-20. (canceled)
 21. A membrane comprising a water-permeable matrix inwhich is included at least two enzymes, urease and carbonic anhydrase,wherein the membrane is configured in a device such that it inhibits gasexchange from a sample to an air space in a region of the membrane. 22.The membrane of claim 21 in which said at least two enzymes areimmobilized in said water-permeable matrix.
 23. The membrane of claim21, which is in physical contact with an ion-selective electrode. 24.The membrane of claim 21, which is in physical contact with a detector.25. The membrane of claim 21, which is physically attached to anion-selective electrode by means of an O-ring.
 26. The membrane of claim21, which is attached to a fixture capable of mating with anion-selective electrode.
 27. The membrane of claim 21, which is formedby applying a liquid mixture onto a surface and allowing said liquidmixture to dry on said surface.
 28. The membrane of claim 27, whichforms a component of a urea detecting device.
 29. The membrane of claim28 in which the urea detecting device is selected from the groupconsisting of an amperometric electrode, a potentiometric ion-selectiveelectrode, an optical sensor, and a conductimetric electrode.
 30. Themembrane of claim 28 in which the urea detecting device comprises amicrofabricated sensor.
 31. The membrane of claim 30 in which themicrofabricated sensor comprises an ion-selective electrode.
 32. Themembrane of claim 30 in which the microfabricated sensor is responsiveto ammonium ions.
 33. The membrane of claim 21 in which saidwater-permeable membrane is latex.
 34. The membrane of claim 21comprising a polymer selected from the group consisting of celluloseacetate, nitrocellulose, polyurethane, bovine serum albumin cross-linkedby glutaraldehyde, acrylamide, cellophane, film-forming latex, andmixtures thereof. 35-54. (canceled)